PROJECT SUMMARY We consider the fundamental issue of whether a level of peptide-MHC recognition in lymph nodes (LN) affects the biology of T cells. This problem is currently under intense analysis in different contexts, all having in common a chronic antigenic exposure: chronic viral infections, responses to cancer, and autoimmunities. Highly discussed now is the concept of ?T cell exhaustion? and what it means transcriptionally and biologically. This proposal is based on human and mouse data demonstrating that insulin is one key autoantigen in the initiation of autoimmune diabetes. Because insulin continuously circulates at low to high pM concentration, we hypothesized that there could be acquisition and presentation of insulin by the antigen presenting cells (APC) of the secondary lymphoid organs. The project is anchored on two findings: i] our previous work showing that interactions between anti-insulin T cells and B cells extend beyond the pancreatic LN to all secondary LNs, resulting in insulin autoantibodies and, ii] new results showing that by 3-4 weeks of age in the NOD mouse there appears to be presentation of insulin in LNs. Thus, even at low level of circulating insulin, there are sufficient peptide/MHC complexes presented in LNs to alter the behavior of autoreactive CD4 T cells. Our three Specific Aims are: 1. Imaging of insulin reactive CD4 T cells; and 2 and 3. The biology and diabetogenicity of insulin reactive CD4 T cells. The behavior of the anti-insulin transgenic T cells will be compared between NOD and NOD mice that express altered insulin epitopes that cannot be detected by the anti-insulin T cells: we create a situation where the insulin reactive T cells can or cannot respond to insulin in LNs. In Aim 1, we visualize the T cells in lymph nodes and examine how their behavior based on the dose of antigen, age of mice, and systemic infection. In Aim 2 and 3, we evaluate how exposure to insulin conditions the diabetogenicity of the insulin reactive T cells, relate the behavior of anti-insulin TCR transgenic to the polyclonal anti-insulin T cell pool, and identify the gene expression patterns that are induced by systemic exposure to autoantigen. Technically, this work relies on a broad armamentarium that includes two-photon microscopy, flow cytometry and cell sorting, antigen presentation assays, cytokine production measurements, diabetes incidence and pathology, and qRT-PCR and RNAseq to dissect the functional consequences of insulin detection by the insulin reactive CD4 T cells. Surprisingly, our early results indicate that the presentation of systemic insulin by the LN and spleen APCs causes the autoreactive pool of T cells that escapes negative selection to be partially activated and become ?poised? to effector function. Our evaluation will show what changes take place in an autoreactive T cells that are chronically exposed to low levels of antigen and will provide new directions to study T cell mediated autoimmunities.